The identification of human immunodeficiency virus type 1 (HIV-1) as the etiological agent of AIDS has led to the proposal of novel intervention strategies to block HIV-1 infection and viral replication. Because HIV-1 infection can be detected early, when the vast majority of the target cells are uninfected, gene therapy is an attractive approach. Our long-ter strategy is to stably transduce pluripotent hematopoietic stem cells with anti-HIV-1 agents that will confer "intracellular immunity" to progeny CD4+ lymphocytes and macrophages, athe permissive host for HIV-1. This approach necessitates; (1) the ability to functionally transduce pluripotent hematopoietic stem cells; and (2) an agent that confers resistance to HIV- 1. We will systematically test the ability of established oncoretroviral and AAV-based gene transfer vectors to functionally transduce human and murine hematopoietic stem cells. We will also examine various well- established methods for gene transfer for their ability to introduce and express transgenes with minimal disruption to cell viability and renewal potential. We will also actively explore the use of HIV-1 pseudotyped vectors. As described in the proposal, these vectors may be advantageous for targetting non-dividing cells., such as pluripotent stem cells. The efficacy of these HIV-based vectors will be assessed in parallel with oncoretroviral and AAV-vectors. Secondly, we will continue to develop the inhibitory potential of two novel peptide inhibitors of the essential HIV-1 regulatory protein, Rev. We have previously identified two novel protein inhibitors of Rev; a new class of dominant-negative Rev mutants and a selected peptide that binds to Rev's effector domain. The potency of these new Rev inhibitors suggest they may be efficacious anti-viral agents. We will perform a series of experiments to directly test their ability to block HIV-1 replication. We will continue to analyze their mechanism of action using biochemical structure- function experiments. These studies will facilitate the development of other potent peptide derivatives.